BackgroundMutations in about 50 genes have been identified as monogenic causes of nephrotic syndrome, a frequent cause of CKD. These genes delineated the pathogenetic pathways and rendered significant insight into podocyte biology.MethodsWe used whole-exome sequencing to identify novel monogenic causes of steroid-resistant nephrotic syndrome (SRNS). We analyzed the functional significance of an SRNS-associated gene in vitro and in podocyte-like Drosophila nephrocytes.ResultsWe identified hemizygous missense mutations in the gene TBC1D8B in five families with nephrotic syndrome. Coimmunoprecipitation assays indicated interactions between TBC1D8B and active forms of RAB11. Silencing TBC1D8B in HEK293T cells increased basal autophagy and exocytosis, two cellular functions that are independently regulated by RAB11. This suggests that TBC1D8B plays a regulatory role by inhibiting endogenous RAB11. Coimmunoprecipitation assays showed TBC1D8B also interacts with the slit diaphragm protein nephrin, and colocalizes with it in immortalized cell lines. Overexpressed murine Tbc1d8b with patient-derived mutations had lower affinity for endogenous RAB11 and nephrin compared with wild-type Tbc1d8b protein. Knockdown of Tbc1d8b in Drosophila impaired function of the podocyte-like nephrocytes, and caused mistrafficking of Sns, the Drosophila ortholog of nephrin. Expression of Rab11 RNAi in nephrocytes entailed defective delivery of slit diaphragm protein to the membrane, whereas RAB11 overexpression revealed a partial phenotypic overlap to Tbc1d8b loss of function.ConclusionsNovel mutations in TBC1D8B are monogenic causes of SRNS. This gene inhibits RAB11. Our findings suggest that RAB11-dependent vesicular nephrin trafficking plays a role in the pathogenesis of nephrotic syndrome.

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Background: Variants in TBC1D8B cause nephrotic syndrome. TBC1D8B is a GTPase-activating protein for Rab11 (RAB11-GAP) that interacts with nephrin,,but how it controls nephrin trafficking or other podocyte functions remains unclear. Methods: We generated a stable deletion in TBC1D8B using microhomology-mediated end joining genome editing. Ex vivo functional assays utilized slit diaphragms in podocyte-like Drosophila nephrocytes. Manipulated endocytic regulators in transgenic mice provided a comprehensive functional analysis of TBC1D8B . . Results: A null allele of Drosophila TBC1D8B exhibited nephrocyte-restricted nephrin mislocalization, similar to patients with isolated nephrotic syndrome who have variants in the gene.. The protein was required for rapid nephrin turnover in nephrocytes and for endocytosis of nephrin induced by excessive Rab5 activity. The protein expressed from TBC1D8B bearing the edited deletion predominantly localized to mature early endosomes and late endosomes and was required for endocytic cargo processing and degradation. Silencing Hrs, a regulator of endosomal maturation, phenocopied loss of TBC1D8B. Low-level expression of murine TBC1D8B rescued loss of the Drosophila gene, indicating evolutionary conservation. Excessive murine TBC1D8B selectively disturbed nephrin dynamics. Finally, we discovered four novel TBC1D8B variants within a cohort of 363 FSGS patients and validated functional impact of two variants in Drosophila, suggesting a personalized platform for TBC1D8B-associated FSGS. Conclusion: Variants in TBC1D8B are not infrequent among FSGS patients. TBC1D8B, functioning in endosomal maturation and degradation, is essential for nephrin trafficking.